plant regeneration of pear leaf mesophyll protoplasts for the cv. Williams' Bon Chretien (also known, commercially, as Bartlett pear). This is the first report of plant ...
Plant Cell Reports
Plant Cell Reports (1988) 7:587-589
© Springer-Verlag 1988
Plant regeneration from mesophyll protoplasts of Williams' Bon Chretien (syn. Bartlett) pear (Pyrus communis L.) S . J . Ochatt and J . B . Power Plant Genetic Manipulation Group, Department of Botany, University of Nottingham, Nottingham, NG7 2RD, United Kingdom Received July 13, 1988/Revised version received August 29, 1988 - Communicated by I. Potrykus
ABSTRACT Leaf protoplasts of axenic shoot cultures of Pyrus communis L. cv. Williams' Bon Chretien (syn. Bartlett) underwent cell wall regeneration and division to give multicellular colonies in a modified Munashige and Skoog medium which lacked ammonium ions, but supplemented with 1-naphthaleneacetic acid (NAA), 4-indole-3yl-acetic acid, 6-benzylaminopurine (BAP) and casein hydrolysate. Protoplast-derived colonies gave callus on Murashige and Skoog salts medium with NAb and BAP and exhibited shoot regeneration on half-strength Murashige andiSkoog medium supplemented wi}h 0.2 mg i" 4-~ndole-3ylrbutyric acid, 2.~ mg i-- BAP, 0.2 mg i-- gibberel~ic acid, 50 mg 1-- casein hydrolysate and 10 mg l-Ca-pantothenate. Following rooting, protoplast-derived plants of pear were transferred to the glasshouse where they completed acclimatization. ABBREVIATIONS BAP, 6-benzylaminopurine; FPE, final plating efficiency; GA., gibberellic acid; IAA, J 4-indole-3yl-acetic acid; IBb, 4-indole-3yl-but yric acid; IPE, initial plating efficiency; NAA, 1-naphthaleneacetic acid; f.wt., fresh weight; MES, 2-N-morpholinoethane sulfonic acid; MS, Murashige and Skoog (1962); %PE, % plating efficiency; PVP-10, polyvinylpyrrolidone (Av. MW 10,000); FDA, fluorescein diacetate. INTRODUCTION Genetic improvement of rosaceous top-fruit tree species based on somatic methods such as somaclonal variation and somatic hybridisation relies on the establishment of reproducible protoplast-to-tree systems (Ochattet al. 1988). Despite their considerable economic importance, plant regeneration from protoplasts of top-fruit trees has only been reported for wild pear (Ochatt and Caso 1986), Colt cherry (Ochattet al. 1987), sour cherry (Ochatt and Power 1988a) and, more recently, apple (?atat-Ochatt et al. 1988). Additionally, protoclonal variation has been described in wild pear rootstock regenerants (Ochatt 1987). The development of protoplast systems for scion varieties of the common pear (P. communis) has been restricted to mesophyll protoplast isolations for the cv. Williams' (Revilla et al. 1987), an enhancement of protoplast division after electroporation (Rech et al. 1987) and the
Offprmtreques~ to: S.J. Ochatt
induction of rhizogenesis in protoplast-derived calli for the cv. Conference (Ochatt and Power 1988b). Plant regeneration from nucellar callus of pear scion varieties was reported by Yehia (1985). This paper describes the isolation, culture and plant regeneration of pear leaf mesophyll protoplasts for the cv. Williams' Bon Chretien (also known, commercially, as Bartlett p e a r ) . This is the first report of plant regeneration from protoplasts of a scion variety in the genus Pyrus. MATERIALS AND METHODS Plant Materials and Protoplast Isolation Shoot cultures of P. communis L. cv. Williams' Bon Chretien (syn. Bartlett) were maintained by monthly subculturing on }gar solidified (0.~% w/v) MS medium with 0.1 mg 1-- IBA and 2.0 mg i-- BAP (pH 5.8), at 25°C with continuous illumination (1000 lux, daylight fluorescent tubes). For protoplast isolation, the most recent fully expanded leaves taken from the axenic shoot cultures were chopped (I-2 mm strips) and plasmolysed (I h) in CPW 13M medium (Power et al. 1984). Leaf tissues (I g f.wt.) were digested in 10 ml of an enzyme mixture which consisted of 1.0% (w/v) Cellulase Onozuka R-10, 0.1% (w/v} Pectolyase Y-23, 5.0 mM MES and 1.0% (w/v) PVP-10, in CPW 13M medium (pH 5.6). Incubation 116 h, 25°C with constant agitation (40 cycles min- )) and protoplast release was as described earlier (Revilla et al. 1987) with continuous illumination (100 lux, cool white fluorescent tubes). Protoplast Culture The absence of a cell wall was confirmed with Calcofluor White (Nagata and Takebe 1970) and viability assessed with FDA (Power et al. 1984). Protoplasts were dil~ted to 0.1, 0.251 0.5, 0.75, 1.0, 2.5 or 5.0 x 10 ~ protoplasts ml-- with either K8P, Km8P (Kao and Michayluk 1975) or MS-based media; the latter modified with or without the inclusion of ammonium ions (Ochatt and Caso 1986; Ochatt and Powe~ 1988b) and casein hydrolysate (0, 50 or 100 mg i-'). All MS-based protoplast media were f u r ~ e r supplemented with 0.0, 0.1, 0.5, 1.0 or 2.0 mg 1 ] NAb and/or IAA and 0.0, 0.1, 0.2, 0.4 or 0.8 mg 1 -] BAP, with 9.0% (w/v) mannitol as osmoticum. Protoplasts were dispensed (4 ml aliquots) into 3.5 cm Petri dishes, sealed with Nescofilm, and maintained (25°C) in the dark or with a continuous illumination of 100 or 400 lux (cool white
588 fluorescent tubes), as e~ther stationary or shaken cultures (40 cycles min- ) until microcalli (I-2 mm diam.) were visible by day 60. During this period, the osmotic pressure of the culture media was progressively reduced by adding an osmoticum-free medium counterpart in.a 3:1 ratio (protoplast: fresh medium) at 10-day intervals and beginning on day 10 of culture. Micro~alli were transferred to MS medium with 2.0 mg i-- NAA, 0.5 mg i, BAP and 0.8% (w/v) agar for further proliferation (over three 2-week passages). Dishes were examined twice weekly and the % PE calculated after day 10 (IPE) and day 60 (FPE). IPE was defined as the percentage of protoplasts that had resynthesized a cell wall and divided at least once, whilst FPE was the percentage of the originally plated protoplasts that had regenerated to microcalli. All experiments involved three replicate dishes ~er treatment and were repeated at least twice. Plant Regeneration Protoplast-derived calli (125 mm 3, ca. 100 day's from protoplast isolation) were transferred for shoot regeneration, to a range of media based on half or full-strength MS s}pplemented'with 0.0, 0.1, 0.2, 0.4 or 0.8 mg 1-] IBA or NAA and 0.0, 0.5, 1.0, 2.0 or 5.0 mg i -~ BAP~ Casein hydrolysate (0, }0 or 100 mg l- ), GA~ (0.0, 0.1, 0.2 or 0.4 mg I-') and Ca-pan}othenat~ (0.0, 2.5, 5.0, 10.0, 25.0 or 50.0 mg i-'), alone or in combination were added to assess their influence on shoot bud regeneration. The percentage of regenerating calli and the number of regenerated shoots per callus were recorded. All such media assessments were replicated at least ten times with all experiments being repeated twice. For multiplication and internode elongation, regenerated pear shoots were transferred to half-~trength MS medium with 2.0 mg 1-- BAP and 0.2 mg l- GAz, for three successive subcultures (4 weeks eacM). Shoots (3.0 cm in height) were rooted after 4 weeks_~ulture in half-strength MS medium with 3.0 mg 1 - IBA. Plants were finally transferred to soil-less compost (Professional Levington M3, Fisons, U,K.i, and each pot was enclosed in a polyethylene bag after watering and maintained in the glasshouse (23 + 5°C; 16 h photoperiod, 10000 lux; cool whi-te f l u o r e s c e n t tubes). Bags were progressively opened over a 3 week period and by 4 weeks ex vitro transfer and acclimatization was considered to be complete. RESULTS High yields [7.7 x 106 g-1 f.wt.) of pear protoplasts were coupled with a viability of 83%. Optimal ~nd sustained division was at a density of 2.5 x 10 J prot6plasts ml- in MS salts m~dium which lacke9 ammonium ions,lbUt with 1.0 mg I~- NAA, 1.0 mg l- IAA, 0.8 mg 1 ~ BAP and 50 mg l- casein hydrolysate, which was an essential media constituent (Table I). Media based on Kao and Michayluk (1975) salts or those MS salts media with ammonium ions supported only cell wall regeneration. Media which lacked both ammonium salts and casein hydrolysate supported initial division, but growth was arrested with associated cell browning and death when colonies had reached the 10-cell stage. Data relating to the effect on division of illumination and agitation, as assessed by PE measurements, is given in Table I. Cell wall regeneration was completed after 6-7 days and first division by 8-10 days. A maximum FPE of 1.89% was achieved after 60 days (Table I). Following proliferation of individual pear
Table I: Effect of casein hydrolysate (mg 1 -I) on % IPE and FPE of Williams' pear mesophyll proto~lasts on ammonium-free MS salt~ with 1.0 mg i-- NAA, 1.0 mg l- IAA and 0.8 mg l- BAP maintained under different cultural conditions.
casein hydrolysate (mg i -I) 0
Protoplast culture
50
100
cohditions IPE
FPE
IPE
FPE
IPE
FPE
0.00 0.00
Dark
stationary shaken
0.00 0.00
0.00 0.00
